Optimization of porous glass chromatography for size-fractionation of bovine casein micelles

Permeation chromatography on porous glass was carried out with skim milk at 20 or 30 °C using CPG-10 (50 nm) or a dual column system of CPG-10 (50 nm) followed by CPG-10 (300 nm). On columns of CPG-10 (50 nm) casein micelles were eluted at the void volume and were rapidly and efficiently resolved from non-micellar protein without micelle dissociation. The dual column system resulted in the additional resolution of the micelles into different size ranges. Examination of the resolved micelle fractions by electron microscopy showed a gradual decrease of weight average diameter (Dw) from 228·4 nm in the void volume fraction to 86·3 nm in the smallest micelle fraction. The translucent upper layer of a micelle sediment obtained by ultracentrifugation of skim milk at 30 °C consisted of casein aggregates intermediate in size between monomeric protein and the bulk micelle fraction as shown by its elution behaviour on CPG-10 (50 nm). These aggregates were enriched more than 2-fold with κ-casein relative to skim milk, were devoid of αs2-casein and had an estimated value of Dw of 33 nm. The ultracentrifugate serum contained ∼ 2·5% of total milk casein which had the elution characteristics of monomeric protein on CPG-10 (50 nm). It was concluded that the translucent sediment consisted of the smallest micelle fraction of skim milk and represented the minimum size range for casein polymerization in the natural milk environment. Overall, the results show that porous glass chromatography is an effective and convenient tool for the isolation and study of casein micelles.